Agent for inducing interferon production containing lactic acid bacteria

ABSTRACT

This invention provides an IFN inducer comprising, as an active ingredient, lactic acid bacteria and capable of inducing IFN production, an immunopotentiating agent or prophylactic agent against virus infection comprising such inducer, and a food or drink product comprising such IFN inducer and having IFN-inducing activity, immunopotentiating activity, or prophylactic activity against virus infection. The agent for inducing IFN production comprises, as active ingredients, lactic acid bacteria that can activate plasmacytoid dendritic cells (pDCs) and promote IFN production, such as  Lactococcus garvieae  NBRC100934,  Lactococcus lactis  subsp.  cremoris  JCM16167,  Lactococcus lactis  subsp.  cremoris  NBRC100676,  Lactococcus lactis  subsp.  hordniae  JCM1180,  Lactococcus lactis  subsp.  hordniae  JCM11040,  Lactococcus lactis  subsp.  lactis  NBRC12007,  Lactococcus lactis  subsp.  lactis  NRIC1150,  Lactococcus lactis  subsp.  lactis  JCM5805,  Lactococcus lactis  subsp.  lactis  JCM20101,  Leuconostoc lactis  NBRC12455,  Leuconostoc lactis  NRIC1540,  Pediococcus damnosus  JCM5886, or  Streptococcus thermophilus  TA-45.

TECHNICAL FIELD

The present invention relates to an agent for inducing interferonproduction containing lactic acid bacteria and a pharmaceutical productand a food or drink product containing the agent for inducing interferonproduction.

BACKGROUND ART

Bacteria, including lactic acid bacteria, are recognized and englobed bya group of immunocytes referred to as the innate immune system, and theycause physiological reactions, such as cytokine or chemokine production,changes in gene expression, and epigenetic gene modification. Cells ofthe innate immune system can be roughly classified as macrophages,natural killer (NK) cells, and dendritic cells. In contrast to thelong-term antigen-specific reactions of the acquired immune system, thereactions of the innate immune system are short-lasting,non-antigen-specific, inclusive reactions. The innate immune systemplays a key role in primary responses against infection with bacteria orviruses, and, in particular, dendritic cells are potent and criticalconstitutive cells. Dendritic cells are highly flexible, a great numberof different subspecies thereof exist, and these cells can be roughlyclassified into myeloid dendritic cells (mDCs), CD8+ dendritic cells(CD8+ DCs), and plasmacytoid dendritic cells (pDCs). mDCs mainly releaseinflammatory cytokines, such as interleukin-12 (IL-12) and tumornecrosis factor-α (TNF-α), upon infection with bacteria and induceactivation of helper T cells (CD4+ T cells). CD8+ DCs are high-powercells producing IL-12, which play a key role in induction of cytotoxic Tlymphocytes (CTLs) upon virus infection or cross-priming of cancerantigens. pDCs are major cells producing type I interferon (IFN)exhibiting growth-inhibiting activity against viruses in vivo, and theyplay a critical role in antiviral biophylaxis. Representative examplesof type I interferon include IFN-α and IFN-β. In order to induce suchsubstances, it is necessary to stimulate Toll-like receptors (TLRs), inparticular, endosomal TLRs, such as TLR3, TLR7, or TLR9. In general,viral double-stranded RNA, viral single-stranded RNA or an antiviralagent (imidazoquinoline), and non-methylated CpG DNA comprising cytosineand guanine joined by a phosphodiester bond are known as a TLR3 ligand,a TLR7 ligand, and a TLR9 ligand, respectively. Thus, nucleic acids ofbacteria or viruses are known to serve as ligands in the induction oftype I interferon production. IFN-α has been put into practical use as atherapeutic agent for hepatitis B, hepatitis C, chronic myeloidleukemia, multiple myeloma, renal cancer, and other diseases, and IFN-βhas been put into practical use as a therapeutic agent for multiplesclerosis, in addition to hepatitis B and hepatitis C. Accordingly, pDCis considered to be the most important cell from the viewpoint ofbiophylaxis, and antiviral prophylaxis, in particular IFN-γ is cytokinethat is classified as type II interferon, and it is mainly produced byNK or Th1 cells, although antiviral effects thereof are weak.Accordingly, a main function thereof is considered to be enhancement ofantiviral effects of IFN-α and IFN-β. In addition, the most recentlydiscovered IFN-λ is classified as type III interferon, such a cytokinehas drawn attention recently because of its potent antiviral effectsverified in recent years. IFN-λ is produced mainly by pDCs in anorganism, as with the case of type I IFN.

In addition to viruses, a certain bacteria are known to activate pDC orproduce IFN-α. As bacteria that are verified to activate pDC, one of thefood-poisoning bacteria, Staphylococcus aureus has been reported. Asbacteria that enhance IFN-α production in the blood, pathogenicbacteria, such as Chlamydia, Salmonella, Mycobacteria, and Listeria, areknown. While some lactic acid bacteria have been reported to enhanceIFN-α production (see Non-Patent Documents 1 and 2), the correlationthereof with pDC remains unknown, and screening has never been conductedusing the capacity for IFN-α production or pDC activation as theindicator. Also, lactic acid bacteria have been reported to enhanceIFN-β production (see Patent Documents 1 and 2 and Non-Patent Document3) and to enhance IFN-γ production (see Patent Document 3); however, thecorrelation thereof with pDC also remains unknown. While it has beenreported that IFN-λ has antiviral activity (see Non-Patent Documents 4and 5) and IFN-λ is mainly produced by pDCs (Non-Patent Document 6), thecorrelation of IFN-λ with lactic acid bacteria remains unknown.

PRIOR ART DOCUMENTS Patent Documents

-   Patent Document 1: JP Patent Publication (Saihyo) No. 2009-005123 A-   Patent Document 2: JP Patent Publication (Saihyo) No. 2009-005124 A-   Patent Document 3: JP Patent Publication (Kokai) No. 2006-028047 A-   Patent Document 4: JP Patent Publication (Kokai) No. 2001-46020 A-   Patent Document 5: JP Patent Publication (Kokai) No. 2000-262247 A

Non-Patent Documents

-   Non-Patent Document 1: Yuichiro Fukui, Nobuhiro Yajima: Abstracts    for the Annual Meeting of the Japanese Association for Food    Immunology, 2006 (Oct. 23 to 24, 2006) Non-Patent Document 2:    “Shokuhin to Kaihatsu (Food Processing and Ingredients),” Vol. 42,    NO. 5, 2007, pp. 85-87-   Non-Patent Document 3: Tadaomi Kawashima, Ikuko Nishimura: Abstracts    for the Annual Meeting of the Japan Society for Lactic Acid    Bacteria, 2010 (Jul. 26 to 27, 2010)-   Non-Patent Document 4: Nature Immunology, 4: 69-77, 2002-   Non-Patent Document 5: Gastroenterology, 131: 1887-1898, 2006-   Non-Patent Document 6: Blood, 115: 4185-90, 2010-   Non-Patent Document 7: The Journal of Immunology, 186: 1685-1693,    2011-   Non-Patent Document 8: Journal of Leukocyte Biology 83: 296-304,    2008

SUMMARY OF THE INVENTION Object to Be Attained by the Invention

The present invention is intended to provide an IFN inducer capable ofinducing IFN production comprising, as an active ingredient, lactic acidbacteria, an immunopotentiating agent or prophylactic agent againstvirus infection comprising such inducer, and a food or drink productcomprising such inducer and having IFN inducing activity,immunopotentiating activity, or prophylactic activity against virusinfection.

Means for Attaining the Object

The present inventors constructed an assay system involving the use ofpDC activation as the indicator and selected lactic acid bacteria thatwould potentiate the prophylactic activity against virus infectionthrough ingestion thereof.

As a result, they discovered that some spherical-shaped lactic acidbacteria would activate pDCs and induce interferon production from suchpDCs. They further discovered that some lactic acid bacteria would becapable of exerting the effects in an organism even they were orallyadministered. The present inventors discovered that the lactic acidbacteria could be used as agents for inducing IFN production, and suchbacteria could also be used for prophylaxis against virus infectionbecause of the immunopotentiating activity of an organism. This has ledto the completion of the present invention.

Specifically, the present invention is as follows.

[1] An agent for inducing IFN production comprising, as an activeingredient, lactic acid bacteria capable of activating plasmacytoiddendritic cells (pDCs) and inducing IFN production or a cultured productthereof.

[2] The agent for inducing IFN production according to [1], wherein theprocessed product of lactic acid bacteria is a fraction containingnucleic acids.

[3] The agent for inducing IFN production according to [1] or [2],wherein IFN is type I IFN or type III IFN.

[4] The agent for inducing IFN production according to any of [1] to[3], wherein IFN is at least one member selected from the groupconsisting of IFN-α, IFN-β, and IFN-λ.

[5] The agent for inducing IFN production according to any of [1] to[4], wherein, when the agent is orally administered, the lactic acidbacteria capable of activating plasmacytoid dendritic cells (pDCs) andinducing IFN production are highly tolerant to the gastric juice orintestinal juice and are capable of reaching the intestinal canal alive.

[6] The agent for inducing IFN production according to [5], wherein thelactic acid bacteria capable of activating plasmacytoid dendritic cells(pDCs) and inducing IFN production are Lactococcus lactis subsp. lactisJCM5805.

[7] An immunopotentiating agent comprising the agent for inducing IFNproduction according to any of [1] to [6].

[8] An agent for prevention or treatment of virus infection comprisingthe agent for inducing IFN production according to any of [1] to [6].

[9] The immunopotentiating agent according to [7], which is an oralpreparation.

[10] The agent for prevention or treatment of virus infection accordingto [8], which is an oral preparation.

[11] A food or drink product comprising the agent for inducing IFNproduction according to any of [1] to [6].

[12] The food or drink product according to [11], which is cheese oryogurt.

[13] A method of screening for lactic acid bacteria capable ofactivating plasmacytoid dendritic cells (pDCs) and inducing IFNproduction comprising culturing the lactic acid bacteria with bonemarrow cells and detecting activation of plasmacytoid dendritic cells(pDCs) and induction of IFN production,

wherein, when plasmacytoid dendritic cells (pDCs) are activated and IFNproduction is induced, the lactic acid bacteria are determined to becapable of activating plasmacytoid dendritic cells (pDCs) and inducingIFN production.

[14] A host microorganism for a recombinant vaccine comprising the agentfor inducing IFN production according to any of [1] to [6].

This description includes part or all of the content as disclosed in thedescription and/or drawings of Japanese Patent Application No.2010-293810, which is a priority document of the present application.

Effects of the Invention

As described in the examples of the present application, the agent forinducing IFN production comprising, as an active ingredient, theparticular lactic acid bacteria of the present invention is capable ofactivating pDC and inducing production of interferon, such as IFN-α, invitro and in vivo. As a result of induction of interferon production invivo, immune responses of an organism are potentiated, the organism isprotected from infection with viruses or the like, and virus infectionscould be treated. The IFN inducer comprising, as an active ingredient,lactic acid bacteria described above can be used as a pharmaceuticalproduct for potentiating immune responses for prevention or treatment ofvirus infection. Further, such inducer can be used as a component of afood or drink product for potentiating immune responses useful forprevention of virus infection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a list of the tested lactic acid bacteria (Part 1).

FIG. 1B shows a list of the tested lactic acid bacteria (Part 2).

FIG. 1C shows a list of the tested lactic acid bacteria (Part 3).

FIG. 2A shows the number and ratio of lactic acid bacteria producing 50pg/ml or more IFN-α, when compared rod-shaped bacteria withspherical-shaped bacteria.

FIG. 2B shows the number and ratio of lactic acid bacteria producing 100pg/ml or more IFN-α, when compared rod-shaped bacteria withspherical-shaped bacteria.

FIG. 3 shows electron microscopy of Lactococcus lactis JCM5805 (FIG. 3A)and Lactococcus lactis JCM20101 (FIG. 3B).

FIG. 4 shows differences in lactic acid bacteria recognition (uptake) ofpDCs. FIG. 4A, FIG. 4B, and FIG. 4C show Lactococcus lactis JCM5805,Lactococcus lactis JCM21101, and Lactobacillus rhamnosus ATCC53103,respectively.

FIG. 5A shows the amount of IFN-α production from various lactic acidbacteria.

FIG. 5B shows the amount of IFN-β production from various lactic acidbacteria.

FIG. 5C shows the amount of IFN-γ production from various lactic acidbacteria.

FIG. 5D shows the amount of IFN-λ production from various lactic acidbacteria.

FIG. 6A shows the capacity for pDC activation of lactic acid bacteriahaving the capacity for inducing IFN-α production and the expressionlevels of MHCI, CD40, CD80, and CD86.

FIG. 6B shows the capacity for pDC activation of lactic acid bacteriahaving the capacity for inducing IFN-α production and the expressionlevels of OX40L, PDL-1, and ICOS-L.

FIG. 7 shows the capacity for stimulating IFN-α production of lacticacid bacteria in the presence of either or both pDCs and mDCs.

FIG. 8 shows photographs showing pDC configurations when Lactococcuslactis JCM5805, Lactococcus lactis JCM20101, and Lactobacillus rhamnosusATCC53103 are added to the pDC monoculture system. FIG. 8A shows theresults concerning the control (to which no lactic acid bacteria wereadded), FIG. 8B shows pDCs when Lactococcus lactis JCM5805 is added,FIG. 8C shows pDCs when Lactococcus lactis JCM20101 is added, and FIG.8D shows pDCs when Lactobacillus rhamnosus ATCC53103 is added.

FIG. 9A shows the amounts of IFN-α production when Lactococcus lactisJCM5805, Lactococcus lactis JCM20101, and Lactobacillus rhamnosusATCC53103 are added to the pDC/mDC cells generated from TLR2 and TLR4knockout mice.

FIG. 9B shows the amounts of IFN-α production when Lactococcus lactisJCM5805, Lactococcus lactis JCM20101, and Lactobacillus rhamnosusATCC53103 are added to the pDC/mDC cells generated from TLR7, TLR9, andMyD88 knockout mice.

FIG. 10 shows the capacity for IFN-α activation of DNAs and RNAs ofLactococcus lactis JCM5805 and Lactococcus lactis JCM20101.

FIG. 11 shows a summary of the experimental design examining the effectsof ingestion of Lactococcus lactis JCM5805 using healthy mice.

FIG. 12 shows changes in blood IFN-α levels of healthy mice that hadingested Lactococcus lactis JCM5805.

FIG. 13A shows changes in MHC class II levels in pDCs of the spleen ofhealthy mice that had ingested Lactococcus lactis JCM5805.

FIG. 13B shows changes in MHC class II levels in pDCs of the mesentericlymph nodes of healthy mice that had ingested Lactococcus lactisJCM5805.

FIG. 13C shows changes in CD86 levels in pDCs of the spleen of healthymice that had ingested Lactococcus lactis JCM5805.

FIG. 13D shows changes in CD86 levels in pDCs of the mesenteric lymphnodes of healthy mice that had ingested Lactococcus lactis JCM5805.

FIG. 14 shows a summary of a method for evaluation of Lactococcus lactisJCM5805 using the immunosuppression models.

FIG. 15A shows the effects of ingestion of Lactococcus lactis JCM5805using the immunosuppression models with reference to changes in the bodyweights of mouse models.

FIG. 15B shows the effects of ingestion of Lactococcus lactis JCM5805using the immunosuppression models with reference to changes in theblood IFN-α levels of mouse models.

FIG. 16A shows changes in MHC class II levels in pDCs of theimmunosuppression mouse models that had ingested Lactococcus lactisJCM5805.

FIG. 16B shows changes in CD86 levels in pDCs of the immunosuppressionmouse models that had ingested Lactococcus lactis JCM5805.

FIG. 16C shows the percentage of pDCs in the immunosuppression mousemodels that had ingested Lactococcus lactis JCM5805.

FIG. 16D shows the results of flow cytometric analysis using lymphocytesof the immunosuppression mouse models that had ingested Lactococcuslactis JCM5805.

FIG. 17 shows Lactococcus lactis JCM5805 and a strain equivalent thereto(a strain derived from Lactococcus lactis JCM5805 and a strain fromwhich Lactococcus lactis JCM5805 is derived).

FIG. 18 shows the capacity of live Lactococcus lactis JCM5805,Lactococcus lactis JCM20101, and Lactobacillus rhamnosus ATCC53103 forIFN-α activation.

FIG. 19A shows the percentage of pDCs determined by flow cytometricanalysis of the purity of human pDCs isolated with MACS.

FIG. 19B shows the amount of IFN-α production detected via ELISA whenLactococcus lactis JCM5805 is added to human pDCs isolated with MACS.

FIG. 19C shows IFN-α1, IFN-β, IFN-λ1, and GAPDH gene expression detectedvia RT-PCR when Lactococcus lactis JCM5805 is added to human pDCsisolated with MACS.

FIG. 20A shows a comparison of changes in MHC class II activity in pDCsof the group subjected to ingestion of yogurt containing Lactococcuslactis JCM5805 and of the placebo group.

FIG. 20B shows a comparison of changes in MHC class II activity in pDCsof a subject having high MHC class II activity in the group subjected toingestion of yogurt containing Lactococcus lactis JCM5805 and in theplacebo group.

FIG. 20C shows a comparison of changes in MHC class II activity in pDCsof a subject having low MHC class II activity in the group subjected toingestion of yogurt containing Lactococcus lactis JCM5805 and in theplacebo group.

FIG. 20D shows a comparison of changes in CD86 activity in pDCs of thegroup subjected to ingestion of yogurt containing Lactococcus lactisJCM5805 and of the placebo group.

FIG. 20E shows a comparison of changes in CD86 activity in pDCs of asubject having high MHC class II activity in the group subjected toingestion of yogurt containing Lactococcus lactis JCM5805 and in theplacebo group.

FIG. 20F shows a comparison of changes in CD86 activity in pDCs of asubject having low MHC class II activity in the group subjected toingestion of yogurt containing Lactococcus lactis JCM5805 and in theplacebo group.

FIG. 21 shows a comparison of amounts of IFN-α1 gene transcription inPBMCs compared at week 0 and week 4 after the initiation of the test ofa subject having low MHC class II activity in the group subjected toingestion of yogurt containing Lactococcus lactis JCM5805 and in theplacebo group.

FIG. 22 shows a comparison of amounts of IFN-α production influenced byCpG stimulation in PBMCs compared at week 0 and week 4 after theinitiation of the test of a subject having low MHC class II activity inthe group subjected to ingestion of yogurt containing Lactococcus lactisJCM5805 and in the placebo group.

FIG. 23 shows the results of comparison between the group subjected toingestion of yogurt containing Lactococcus lactis JCM5805 and theplacebo group regarding the development of cold symptoms during theperiod of test product ingestion examined every week.

EMBODIMENTS FOR CARRYING OUT THE INVENTION

Hereafter, the present invention is described in detail.

The present invention relates to an agent for inducing IFN productioncomprising, as an active ingredient, lactic acid bacteria. The term“inducing (or variations thereof) IFN production” used herein refers toinduction of IFN production in vitro and in vivo.

Lactic acid bacteria that can be used as the agent for inducing IFNproduction in the present invention are capable of activatingplasmacytoid dendritic cells (pDCs) and promoting IFN production ofpDCs. Further, lactic acid bacteria that can be used as the agent forinducing IFN production in the present invention are capable ofpromoting expression of activation markers, such as CD80, CD86, and MHCclass II, in pDCs. Whether or not candidate lactic acid bacteria havesuch properties may be determined by, for example, culturing candidatelactic acid bacteria in the presence of bone marrow cells generated frommammalians, such as mice, and detecting the occurrence of pDC activationand induction of production of IFN, such as IFNα and IFNβ. IFN may beassayed by measuring the IFN concentration in a culture solution via,for example, ELISA. Lactic acid bacteria that can be used as an agentfor inducing IFN production in the present invention has property asdescribed below. For example, mouse bone marrow cells from whicherythrocytes have been removed are suspended to a concentration of 5×10⁵cells/ml in RPMI medium (SIGMA) containing 10% FCS and 2 μMβ-mercaptoethanol, Flt-3L is added as a pDC inducing cytokine to a finalconcentration of 100 ng/ml to the resulting cell suspension, theresultant is cultured in a CO₂ incubator at 37° C. in the presence of 5%CO₂, lactic acid bacteria are added thereto 7 days later at 10 μg/ml,the culture supernatant is collected 48 hours later, the IFN-αconcentration in the culture supernatant is assayed via ELISA with theuse of the IFN-α assay kit (PBL), and the determined IFN-α concentrationis preferably 50 pg/ml or higher, and more preferably 100 pg/ml orhigher.

Examples of preferable lactic acid bacteria that can activateplasmacytoid dendritic cells (pDCs) and promote IFN production of pDCsinclude spherical-shaped lactic acid bacteria. Specifically,spherical-shaped lactic acid bacteria that belong to the generaLactococcus, Leuconostoc, Pediococcus, and Streptococcus are morepreferable. Particularly preferable strains are Lactococcus garvieae,Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis,Lactococcus lactis subsp. hordniae, Leuconostoc lactis, Pediococcusdamnosus, and Streptococcus thermophiles.

Specific examples of such lactic acid bacteria include Lactococcusgarvieae NBRC100934, Lactococcus lactis subsp. cremoris JCM16167,Lactococcus lactis subsp. cremoris NBRC100676, Lactococcus lactis subsp.hordniae JCM1180, Lactococcus lactis subsp. hordniae JCM11040,Lactococcus lactis subsp. lactis NBRC12007, Lactococcus lactis subsp.lactis NRIC1150, Lactococcus lactis subsp. lactis JCM5805, Lactococcuslactis subsp. lactis JCM20101, Leuconostoc lactis NBRC12455, Leuconostoclactis NRIC1540, Pediococcus damnosus JCM5886, and Streptococcusthermophilus TA-45. Among these strains, the capacity for inducing IFN-αproduction of Lactococcus lactis subsp. lactis JCM5805 and that ofLactococcus lactis subsp. lactis JCM20101 are particularly high. Thus,use of Lactococcus lactis JCM5805 is particularly preferable.

Lactic acid bacteria that can be used as the agent for inducing IFNproduction in the present invention preferably exert activity of IFNinduction in an organism when such bacteria are ingested orally. Suchlactic acid bacteria are highly tolerant to the gastric or intestinaljuice. For example, such lactic acid bacteria have high-level toleranceto acids and are capable of reaching the intestinal canal alive. WhenLactococcus lactis JCM5805 described above is orally ingested, it iscapable of exerting a significant degree of activity for inducing IFNproduction in an organism.

The lactic acid bacteria described above can be obtained from the RIKENBioResource Center (3-1-1 Koyadai, Tsukuba-shi, Ibaraki, Japan), theBiological Resource Center (NBRC) at the National Institute ofTechnology and Evaluation (http://www.nbrc.nite.go.jp), the CultureCollection Center, Tokyo University of Agriculture(http://nodaiweb.university.jp/nric/), and DANISCO. In addition,bacterial strains equivalent to the strains, such as Lactococcusgarvieae NBRC100934, Lactococcus lactis subsp. cremoris JCM16167,Lactococcus lactis subsp. cremoris NBRC100676, Lactococcus lactis subsp.hordniae JCM1180, Lactococcus lactis subsp. hordniae JCM11040,Lactococcus lactis subsp. lactis NBRC12007, Lactococcus lactis subsp,lactis NRIC1150, Lactococcus lactis subsp. lactis JCM5805, Lactococcuslactis subsp. lactis JCM20101, Leuconostoc lactis NBRC12455, Leuconostoclactis NRIC1540, Pediococcus damnosus JCM5886, or Streptococcusthermophilus TA-45, can also be used. Equivalent strains include strainsderived from the bacterial strains mentioned above, the bacterialstrains from which the strains mentioned above are derived, or offspringstrains of such bacterial strains. Equivalent strains may be conservedin other institutions for culture collection. FIG. 17 shows bacterialstrains derived from Lactococcus lactis JCM5805 and bacterial strainsfrom which Lactococcus lactis JCM5805 is derived. Bacterial strainsequivalent to Lactococcus lactis JCM5805 shown in FIG. 17 can also beused as active ingredients of the agent for inducing IFN production ofthe present invention. The term “Lactococcus lactis JCM5805” used hereinalso refers to such equivalent strains. Other lactic acid bacteria thatcan be used as the agent for inducing IFN production of the presentinvention can be obtained from, for example, the RIKEN BioResourceCenter, American type culture collection (U.S.A.), the Institute forFermentation (2-17-85 Jusohonmachi, Yodogawa Ward, Osaka, OsakaPrefecture, Japan), or the Culture Collection Center, Tokyo Universityof Agriculture (1-1-1, Sakuragaoka, Setagaya, Tokyo, Japan).

The agent for inducing IFN production of the present invention caninduce any of type I interferon (type I IFN), type II interferon (typeII IFN), or type III interferon (type III IFN). Type I IFN is a cytokinethat is effective against virus infection, and examples thereof includeIFN-α1, IFN-α2, IFN-α4, IFN-α5, IFN-α6, IFN-α7, IFN-α8, IFN-α10,IFN-α13, IFN-α14, IFN-α16, IFN-α17, IFN-α21, and IFN-β. An example oftype II IFN is IFN-γ, and an example of type III IFN is IFN-λ. The agentfor inducing IFN production of the present invention has activity ofinducing production of type I IFN, in particular. The agent for inducingIFN production of the present invention activates plasmacytoid dendriticcells (pDCs). When a plasmacytoid dendritic cell is activated, a cellprocess, which is characteristic of the activated dendritic cell,appears, and type I IFN and type III IFN are produced. At this time,lactic acid bacteria, which are active ingredients of the agent forinducing IFN production of the present invention, are incorporated intopDCs. The agent for inducing IFN production of the present invention hasthe high capacity for inducing production of type I IFN and type IIIIFN, and the capacity for inducing production of IFN-α, which is type IIFN, is particularly high. The agent for inducing IFN production of thepresent invention is also capable of inducing production of type II IFN,such as IFN-γ, from NK cells or Th1 cells. Immune activity of anorganism is enhanced via induction of IFN production. However, lacticacid bacteria, which are active ingredients of the agent for inducingIFN production of the present invention, are capable of inducing theexpression of PDL-1. PDL-1 is a programmed death ligand-1 (PD-1), andPDL-1 binds to PD-1 and induces regulatory T cells, so that PDL-1 canprevent an immune system from being excessively activated and suppressthe autoimmune reactions. Specifically, the agent for inducing IFNproduction of the present invention is not only capable of inducing IFNproduction and activating the immune functions of an organism but alsocapable of suppressing excessive immune reactions and maintaining thebalanced immune reactions in the organism.

The agent for inducing IFN production of the present invention cansimultaneously induce production of type I IFN and type III IFN.Specifically, production of IFN-α, IFN-β, and IFN-λ can be inducedsimultaneously.

The agent for inducing IFN production of the present invention promotesexpression of CD80, CD86, and MHC class II in pDCs. TLR9 is involved inthe induction of IFN production as a receptor.

The agent for inducing IFN production of the present invention containsa culture product of the lactic acid bacteria above. The term “cultureproduct” refers to live bacteria, killed bacteria, fragmented live orkilled bacteria, lyophilized live or killed bacteria, or a fragmentedproduct, culture solution, or culture extract of such lyophilizedbacteria. The term also refers to part of lactic acid bacteria ortreated lactic acid bacteria. Examples of treated lactic acid bacteriainclude products resulting from enzyme or thermal treatment of lacticacid bacteria and products recovered through ethanol precipitation ofthe products of enzyme or thermal treatment. Further, DNA or RNA of theabove lactic acid bacteria is within the scope of the culture product oflactic acid bacteria. DNA or RNA of the above lactic acid bacteria isconsidered to be capable of activating pDCs and inducing IFN production.

Lactic acid bacteria can be cultured in accordance with a conventionaltechnique using conventional media. Examples of media that can be usedinclude MRS, GAM, and LM17 media, and inorganic salts, vitamins, aminoacids, antibiotics, sera, or other substances may be added thereto,according to need. Culture may be carried out at 25° C. to 40° C. forseveral hours to several days.

After culture, lactic acid bacteria are harvested via centrifugation,filtration, or other means. When used in the form of killed bacteria,bacteria may be sterilized and inactivated with the use of an autoclave,for example.

Activity for inducing IFN production of lactic acid bacteria that can beused as active ingredients of the agent for inducing IFN production ofthe present invention can be assayed by culturing candidate bacteria,culturing IFN-producing cells in the presence of the culture productthereof, and detecting an increase in the amount of IFN produced by theIFN-producing cells. Typically, lyophilized bacteria are used. Theweight of bacteria in the lyophilization product is adjusted to 0.1 to 5mg/ml, and the lyophilization product is then cultured with, forexample, bone marrow cells. Origins of bone marrow cells are notparticularly limited, and bone marrow cells derived from humans or bonemarrow cells derived from non-human animals such as mice can be used.When pDCs are activated and IFN production is induced in the bone marrowcells, the lactic acid bacteria can be determined to be usable as activeingredients of the agent for inducing IFN production of the presentinvention. Activation of pDCs may be detected by, for example, assayingpDC activation markers, and examples of activation markers include CD80,CD86, and MHC class II. Such activation markers can be assayed via cellstaining or flow cytometry using antibodies reacting with such markers.Examples of IFN include type I IFNs such as IFN-α and IFN-β, type IIIFNs such as IFN-γ, and type III IFN such as IFN-λ. Among them, type IIFN and type III IFN are preferable, type I IFN is more preferable, andIFN-α is further preferable. Induction of IFN production may be assayedby determining the amount of IFN in a medium in the culture system via,for example, ELISA.

The present invention includes a method for screening for lactic acidbacteria having activity of inducing IFN production and usable as activeingredients of the agent for inducing IFN production of the presentinvention.

The agent for inducing IFN production of the present invention can beused in the form of a pharmaceutical product that induces IFN productionand enhances immune activity of an organism. Specifically, the agent forinducing IFN production can be used in the form of an immunopotentiatingagent or immunostimulator. Such pharmaceutical products can be used forpreventive or therapeutic agents for diseases, which are already knownto be associated with type I IFN. Examples of such diseases include:cancer, including renal cancer, multiple myeloma, chronic myeloidleukemia, hairy cell leukemia, gliosarcoma, medulloblastoma,astroglioma, malignant melanoma, mycosis fungoides, and adult T cellleukemia; virus infection, including subacute sclerosingpanencephalitis, HTLV-1 associated myelopathy, hepatitis B, andhepatitis C; infection with bacteria, such as chlamydia (sexuallytransmitted disease), Mycobacteriaceae (tuberculosis), listeriosis(ichorrhemia), Staphylococcus (food poisoning), and Helicobacter(gastritis); and autoimmune diseases including multiple sclerosis. Thepharmaceutical product is particularly useful as a prophylactic ortherapeutic agent for virus infection. Since the function of inhibitingdifferentiation of osteoblasts into osteoclasts is known as activity oftype I IFN, it can be used as a preventive or therapeutic agent forosteoporosis.

An antigen associated with a particular disease may be expressed inspherical-shaped lactic acid bacteria, which is the IFN inducer of thepresent invention, via genetic engineering, and the resultant may beused as a vaccine. Since the cell wall of lactic acid bacteria canprotect antigens from gastric acid, such bacterial strains expressingforeign antigens are particularly preferable as host organisms of oralvaccines. In general, vaccines are classified as live vaccines,inactivated whole particle vaccines, or split vaccines. However, livevaccines pose a risk of potentiating the virus virulence, inactivatedwhole particle vaccines may evoke side effects because of the presenceof impurities, and split vaccines with the highest safety areproblematic in terms of efficacy. In order to overcome such drawbacks,development of recombinant vaccines selectively expressing targetantigens has been attempted. If the target antigens are expressed in thespherical-shaped lactic acid bacteria having the effects of IFNinduction according to the present invention, effects of an adjuvant canalso be achieved, and it is thus very useful.

Dosage forms of the agent for inducing IFN production of the presentinvention are not particularly limited. Examples include powder,granules, tablets, syrup, injection preparations, drops, powdered drugs,suppositories, suspensions, and ointments. The pharmaceutical product ofthe present invention may be administered orally or parenterally throughintravenous injection, intramuscular injection, subcutaneousadministration, rectal administration, or transdermal administration,with oral administration being preferable. The agent for inducing IFNproduction may contain an excipient, a disintegrator, a binder, alubricant, a colorant, or the like. Examples of excipients includeglucose, lactose, corn starch, and sorbit. Examples of disintegratorsinclude starch, sodium alginate, powdered gelatin, calcium carbonate,calcium citrate, and dextrin. Examples of binders includedimethylcellulose, polyvinyl alcohol, polyvinyl ether, methylcellulose,ethylcellulose, gum Arabic, gelatin, hydroxypropyl cellulose, andpolyvinyl pyrrolidone. Examples of lubricants include talc, magnesiumstearate, polyethylene glycol, and hydrogenated vegetable oil. A dosecan be adequately determined in accordance with the age, body weight, orsexuality of a patient, a type of disease, severity of symptoms, orother conditions. The agent may be administered once or several separatetimes per day, and a culture product may be administered in an amountequivalent to 1×10⁹ to 1×10¹² cells in a single instance. Alternatively,a dose may be 1 to 1,000 mg of lactic acid bacteria.

The agent for inducing IFN production of the present invention can beincorporated into a food or drink product. Thus, the resulting food ordrink product can be used for induction of IFN production,immunopotentiation, immunostimulation, prophylaxis against virusinfection, or other purposes. Target food or drink products are notparticularly limited, provided that active ingredients for induction ofIFN production are not inhibited. Examples include milk, dairy products,beverage, seasonings, alcoholic beverage, agricultural products,processed forest products, confectioneries, breads, cereals, noodles,seafood products, processed livestock products, oils and fats, processedoils and fats, prepared frozen foods, retort foods, ready-to-eat foods,and food materials. Use of fermented dairy products, such as yogurt orcheese, and drinks containing lactic acid bacteria is particularlysuitable. When used in the form of fermented food or drink products,given amounts of killed lactic acid bacteria having activity of inducingIFN production may be added to fermented food or drink products.Alternatively, such lactic acid bacteria may be used as starters toproduce fermented food or drink products.

A fraction containing large quantities of nucleic acids of the lacticacid bacteria of the present invention can also be used as the agent forinducing IFN production. A nucleic acid may be DNA, RNA, or a mixturethereof, with DNA being preferable. Such fraction can be prepared by,for example, enzyme or thermal treatment (Patent Document 4) or recoveryof a precipitate obtained with the aid of ethanol (Patent Document 5).In the present invention, such fraction is referred to as a processedproduct of lactic acid bacteria. With the use of such fraction, a moreeffective health food or drink product with enriched active ingredientscan be provided.

Examples of the food or drink product of the present invention include ahealth food or drink product, a food or drink product for specifiedhealth use, a food or drink product with nutrient function claims, and adietary supplement food or drink product. The term “food or drinkproduct for specified health use” refers to a food or drink product thatis to be ingested for specified healthcare objectives and has a labelingindicating that the objectives can be expected through ingestionthereof. Such food or drink product may be provided with a labelingindicating that, for example, enhancement of body's immune functions,stimulation of body's immune functions, lowering of susceptibility tocolds, enhancement of tolerance to infection with viruses such asinfluenza virus, norovirus, or rotavirus, or cancer prevention.

EXAMPLES

The present invention is described in detail with reference to thefollowing examples, although the present invention is not limited tothese examples.

Example 1 Preparation of Test Lactic Acid Bacteria <Experimental Method>

The lactic acid bacteria shown in FIG. 1A, FIG. 1B, and FIG. 1C weresubjected to thermal treatment to prepare killed lactic acid bacteria.At the outset, the lactic acid bacteria mentioned above were purchasedfrom microbial strain libraries in Japan and abroad. The lactic acidbacteria were obtained from the Japan Collection of Microorganisms (JCM)of the RIKEN BioResource Center, the Culture Collection Center of theInstitute of Fermentation, Osaka (IFO), the NODAI Culture CollectionCenter (NRIC) of Tokyo University of Agriculture, American Type CultureCollection (ATCC, U.S.A.), and DANISCO. A total of 125 strains of 31different species were obtained. Lactic acid bacteria were subjected tostationary culture in MRS, GAM, or LM17 medium at 30° C. or 37° C. for24 to 48 hours. The strains were harvested, washed three times withsterile water, and then disinfected in an autoclave at 100° C. for 30minutes. Thereafter, the strains were lyophilized, and the concentrationwas adjusted to 1 mg/ml with PBS (Takara Bio).

Example 2 Screening for Lactic Acid Bacteria Having Capacity forInducing IFN-α Production

The capacity of the lactic acid bacteria prepared in Example 1 forinducing IFN-α production was evaluated with reference to pDCactivation.

<Experimental Method>

Bone marrow cells were collected from the femoral bones of C57BL/6 micein accordance with a conventional technique, and erythrocytes wereremoved therefrom. Subsequently, the collected bone marrow cells weresuspended to a concentration of 5×10⁵ cells/ml in RPMI medium (SIGMA)containing 10% FCS and 2 μM β-mercaptoethanol. Flt-3L (R&D Systems) wasadded as a pDC inducing cytokine to a final concentration of 100 ng/mlto the resulting cell suspension, and culture was conducted in a CO₂incubator at 37° C. in the presence of 5% CO₂. Various types of lacticacid bacteria were added thereto 7 days later at 10 μg/ml, and theculture supernatant was collected 48 hours later. The culturesupernatant was subjected to ELISA assays with the use of the IFN-αassay kit (PBL).

<Results>

FIG. 2 shows strains evaluated to be capable of producing 50 pg/ml ormore IFN-α via ELISA. Among the 125 tested strains, activity wasobserved in only 13 strains (i.e., Lactococcus garvieae NBRC100934,Lactococcus lactis subsp. cremoris JCM16167, Lactococcus lactis subsp.cremoris NBRC100676, Lactococcus lactis subsp. hordniae JCM1180,Lactococcus lactis subsp. hordniae JCM11040, Lactococcus lactis subsp.lactis NBRC12007, Lactococcus lactis subsp. lactis NRIC1150, Lactococcuslactis subsp. lactis JCM5805, Lactococcus lactis subsp. lactis JCM20101,Leuconostoc lactis NBRC12455, Leuconostoc lactis NRIC1540, Pediococcusdamnosus JCM5886, and Streptococcus thermophiles TA-45). There were only3 strains (i.e., Lactococcus lactis subsp. lactis NRIC1150, Lactococcuslactis subsp. lactis JCM5805, and Lactococcus lactis subsp. lactisJCM20101) that had been evaluated to be capable of producing 100 pg/mlor more IFN. Most bacteria did not have the capacity for inducing IFN-αproduction on pDCs. This indicates that such activity is not universalamong various types of lactic acid bacteria.

The selected 3 strains inducing the production of IFN at a high level(i.e., 100 pg/ml or more IFN) were spherical-shaped bacteria classifiedas Lactococcus lactis subsp. lactis. As shown in FIG. 2A, further, thehit rate of spherical-shaped lactic acid bacteria is 34.29%, which issignificantly higher than that of rod-shaped lactic acid bacteria (i.e.,0.00%). In the case of strains inducing the production of IFN at a highlevel shown in FIG. 2B, the hit rate of spherical-shaped lactic acidbacteria is 8.57%, which is also higher than that of rod-shaped lacticacid bacteria (i.e., 0.00%). This indicates that activity of stimulatingpDCs to induce IFN-α production is characteristic of spherical-shapedlactic acid bacteria. While activity of directly stimulating pDCs byStaphylococcus aureus was reported, the capacity for pDC activation ofingestible bacteria that are harmless to humans was discovered for thefirst time.

The Lactococcus lactis JCM5805 and JCM20101 strains exhibiting thecapacity for inducing IFN-α production at a particularly significantlevel and, as a negative control, the rod-shaped Lactobacillus rhamnosusATCC53103 strain were subjected to the following analysis.

FIG. 3 shows electron micrographs of the Lactococcus lactis JCM5805 andJCM20101 strains. FIG. 3A shows the JCM5805 strain, and FIG. 3B showsthe JCM20101 strain. These strains were oval-shaped bacteria withapproximately 1-μm major axes and 0.5-μm minor axes. Since rod-shapedbacteria generally have approximately 1-μm minor axes and 3-μm majoraxes, it can be said that such bacteria are very small.

Example 3

Differences in Lactic Acid Bacteria Recognition (Uptake) by pDCs

With the use of the Lactococcus lactis JCM5805 and JCM20101 strains,which were found to be pDC-activating lactic acid bacteria in Example 2,and Lactobacillus rhamnosus ATCC53103 as a negative control, anexperiment was carried out to confirm that activity would depend onrecognition of the bacteria by pDCs; i.e., uptake of the bacteria.

<Experimental Method>

In Example 2, bone marrow cells were cultured by laying a micro glasscover slip (Matsunami Glass Ind., Ltd.). Lactococcus lactis JCM5805,Lactococcus lactis JCM20101, and Lactobacillus rhamnosus ATCC53103labeled with FITC(SIGMA) were added thereto, and culture was conductedin a CO₂ incubator at 37° C. in the presence of 5% CO, for 3 hours.Thereafter, the micro glass cover slip was collected. The cells werestained with anti-B220-PE-Cy5.5 (eBiosciencs), allowed to adhere to theglass slides (Matsunami Glass Ind., Ltd.), and then observed under afluorescent microscope (Olympus Corporation).

<Results>

The results are shown in FIG. 4. FIG. 4A, FIG. 4B, and FIG. 4C showLactococcus lactis JCM5805, the Lactococcus lactis JCM21101 strain, andLactobacillus rhamnosus ATCC53103, respectively. B220-positive red cellsare pDCs. In the Lactococcus lactis JCM5805 and JCM20101 strains, uptakeof the lactic acid bacteria stained green into cells is observed,although Lactobacillus rhamnosus ATCC53103 is not incorporated into thecells. Therefore, whether or not activity occurs is considered to dependon the recognition of bacteria by pDCs.

Example 4 Activity of Lactic Acid Bacteria in Terms of Capacity forInducing IFN Production

The capacity of the lactic acid bacteria having the capacity forinducing IFN-α production for producing cytokines of other types wasexamined.

<Experimental Method>

The killed and lyophilized products (10 μg/ml) of Lactococcus lactisJCM5805, Lactococcus lactis JCM20101, and Lactobacillus rhamnosusATCC53103 and, as positive controls, known TLRLs; i.e., Pam3CSK4 (TLR2L,1 μg/ml, InvivoGen), LPS (TLR4L, 5 ng/ml, SIGMA-ALDRICH), and CpG DNA(TLR9L, 0.1 μM, InvivoGen), were added to the pDC/mDC culture systemdescribed in Example 2, and the culture supernatant was recovered 48hours later. The culture supernatant was subjected to ELISA assays usingthe IFN-α assay kit (PBL), the IFN-β assay kit (PBL), the IFN-γ assaykit (BD Pharmingen), and the IL-28/IFN-λ assay kit (eBiosciencs).

<Results>

The results are shown in FIG. 5A to FIG. 5D. FIG. 5A, FIG. 5B, FIG. 5C,and FIG. 5D show the results concerning IFN-α, IFN-β, IFN-γ, and IFN-λ,respectively. As described in Example 2, the capacity for IFN-αproduction was observed in Lactococcus lactis JCM5805 and in Lactococcuslactis JCM20101, and the titer thereof was equivalent to that of CpG DNA(ODN 1585) (i.e., TLR9L, 0.1 μM). Concerning IFN-β, which is also type IIFN, the capacity was observed selectively in the Lactococcus lactisJCM5805 and JCM20101 strains. Concerning IFN-γ, which is type II IFN,all bacterial strains exerted the capacity for inducting production,although the degree thereof varied among bacterial species. ConcerningIFN-γ, which is type III IFN, induction was observed selectively inLactococcus lactis JCM5805 and in Lactococcus lactis JCM20101.

While the level of IFN-λ induced by IFN-stimulated genes (ISG) is knownto be lower than that of IFN-α or IFN-β, IFN-λ is known to potentiateits antiviral effects in coordination with IFN-α (Non-Patent Document5). Since the Lactococcus lactis JCM5805 and JCM20101 strains arecapable of inducing production of all of type I, type II, and type IIIIFNs, these strains are considered to have very strong antiviralactivity.

Example 5 Activity of Lactic Acid Bacteria in Terms of pDC Activation

The capacity of lactic acid bacteria having the capacity for inducingIFN-α production for pDC activation was examined.

<Experimental Method>

The cells cultured in Example 3 were stained for 30 minutes at 4° C.with the use of anti-CD11b-APC-Cy7 antibody (BD Pharmingen),anti-α220-PerCP antibody (BD Pharmingen), and anti-CD11c-PE-Cy7 antibody(eBiosciencs) for pDC gating, anti-MHC class II-FITC antibody(eBiosciencs), anti-CD40-FITC antibody (eBiosciencs), anti-CD80-APCantibody (eBiosciencs), and anti-CD86-APC antibody (eBiosciencs) asindicators for activation, and anti-OX40L-PE antibody (eBiosciencs),anti-PDL-1-PE antibody (eBiosciencs), and anti-ICOS-L-PE antibody(eBiosciencs) as inhibitory markers. The cells were washed and thenanalyzed with the use of FACS Canto II (BD).

<Results>

The results are shown in FIG. 6A and in FIG. 6B. FIG. 6A shows MHCII,CD40, CD80, and CD86 expression levels, and FIG. 6B shows OX40L, PDL-1,and ICOS-L expression levels. The median fluorescent intensity values(MFI) attained without the addition of lactic acid bacteria are shownabove, and the MFI values attained with the addition of lactic acidbacteria are shown below. According to the activation markers,enhancement was observed with the addition of any lactic acid bacteria.The greatest difference between the Lactococcus lactis JCM5805 andJCM20101 strains and the Lactobacillus rhamnosus ATCC53103 strain havingno capacity for inducing IFN-α production is observed in CD28, which isa T cell activity regulatory molecule, and CTLA-4 ligands (i.e., CD80and CD86). Expression levels were significantly enhanced by theLactococcus lactis JCM5805 and JCM20101 strains. According to theinhibitory markers, an increase was observed in the expression levelswith the addition of lactic acid bacteria, as expected. In particular,PDL-1 expression was significantly activated by the Lactococcus lactisJCM5805 and JCM20101 strains.

As described above, pDCs stimulated by the Lactococcus lactis JCM5805and JCM20101 strains produce IFN-α, thereby potentiating the immunesystem. However, the potentiated immune system may cause autoimmunediseases as side effects. PDL-1, which was confirmed to be induced uponstimulation by the Lactococcus lactis JCM5805 and JCM20101 strains inthis test, is known to bind to PD-1 of the T cell to induce a regulatoryT cell. Specifically, the Lactococcus lactis JCM5805 and JCM20101strains are considered to be capable of maintenance of the immune systemin a well-balanced state while refraining from being excessivelyactivated through PDL-1 expression, in addition to potentiation of theimmune system through IFN-α production.

Example 6

Capacity for Stimulating IFN-α Production of Lactic Acid Bacteria in thePresence of Either or Both pDCs and mDCs

In Example 2, the Lactococcus lactis JCM5805 and JCM20101 strains wereselected on the basis of activity for inducing IFN-α production. In theculture system used for assays, myeloid dendritic cells (mDCs) develop,in addition to pDCs (i.e., a so-called pDC/mDC mixed culture system).The pDC-mDC interactions are considered to be critical in an organism.For example, conversion of pDC into mDC upon virus infection has beenreported. Accordingly, the effects of the addition of lactic acidbacteria in the pDC or mDC monoculture system, the pDC/mDC mixed culturesystem, and the mixed culture system in which pDCs were physicallyseparated from mDCs were examined.

<Experimental Method>

pDCs and mDCs induced from bone marrow cells with the aid of Flt-3L weresubjected to mixed culture in the same manner as in Example 2, and pDCswere separated from mDCs using FACS Aria (BD). Subsequently, pDCs andmDCs were subjected to culture at a density of 1×10⁵ cells/ml under theconditions described below: (1) in a monoculture system in which pDCsare separated from mDCs (indicated as pDC or mDC); (2) in a mixedculture system in which pDCs are physically in contact with mDCs(pDC:mDC=1:1); and (3) a pDC/mDC co-culture system in which physicalcontact between pDCs and mDCs is blocked with a semipermeable membrane(indicated as pDC/mDC or mDC/pDC, cells cultured on a semipermeablemembrane and in contact with lactic acid bacteria are shown on theleft). Transwell filter (Corning) was used as a semipermeable membrane,and the amount of lactic acid bacteria added was 10 μg/ml. Culture wasconducted for 2 days and the amount of IFN-α in the culture supernatantwas then assayed. As a positive control, CpG DNA (ODN1585), the capacityof which for pDC activation has been known, was used at 0.1 μM. SortedpDCs were attached to glass slides (Matsunami glass Ind., Ltd) usingCytospin (Thermo Scientific), stained with Diff-Quick (Sysmex), and thenobserved under a microscope (Olympus Corporation).

<Results>

The results are shown in FIG. 7. The Lactococcus lactis JCM5805 andJCM20101 strains exhibited similar responses. Specifically, IFN-αproduction did not take place in the mDC monoculture system, a smallquantity of IFN-α was induced in the pDC monoculture system, and asignificant level of IFN-α production was observed in the pDC/mDC mixedculture system. FIG. 8 shows photographs showing the configurations ofpDCs when Lactococcus lactis JCM5805, Lactococcus lactis JCM20101, andLactobacillus rhamnosus ATCC53103 are added to the pDC monoculturesystem. FIG. 8A shows the results of a control (without the addition oflactic acid bacteria), FIG. 8B shows pDCs when JCM5805 was added, FIG.8C shows pDCs when JCM20101 was added, and FIG. 8D shows pDCs whenATCC53103 was added. As a result of a comparison of pDCs upon theaddition of the Lactococcus lactis JCM5805 and JCM20101 strains and pDCswithout the addition thereof, a cell process characteristic of anactivated dendritic cell was apparently observed when the Lactococcuslactis JCM5805 and JCM20101 strains were added. When Lactobacillusrhamnosus ATCC53103 was added, however, a process as observed when theLactococcus lactis JCM5805 or JCM20101 strain was added was notdetected. More interestingly, the level of IFN-α production wasdrastically reduced to a level equivalent to that attained in the pDCmonoculture when physical contact between pDCs and mDCs was blocked witha semipermeable membrane. Thus, it was found that the presence of mDCswould be necessary in order to fully induce IFN-α production viaactivation of pDC, although pDCs are primary targets of lactic acidbacteria. In addition, the mDC/pDC cross-talk was found to be mediatedby a cell-to-cell contact instead of a humoral factor. Sincesubstantially the same phenomenon is observed with the addition of CpGDNA, it was verified that a role of mDC in such pDC activation would bea universal mechanism that would not be limited to a particularsubstance (i.e., lactic acid bacteria). Both pDCs and mDCs are presentin a human body, and such mechanism would serve as a key factor whenconsidering application thereof to humans.

Example 7 Identification of Receptors Involved

Signals essential for the Lactococcus lactis JCM5805 and JCM20101strains to produce IFN-α were examined using TLR knockout mice.

<Experimental Method>

TLR2-, TLR4-, TLR7-, TLR9-, and MyD88-knockout mice (8- to 10-week-old,male) and wild-type C57BL/6 mice (8- to 10-week-old, male) werepurchased from Charles River Laboratories. pDCs and mDCs were inducedfrom bone marrow cells of such mice in the same manner as in Example 2,and Lactococcus lactis JCM5805, Lactococcus lactis JCM20101, andLactobacillus rhamnosus ATCC53103 were added. In addition to the 3 TLRLsdescribed in Example 3, TLR7L (ssRNA40, 5 μg/ml, InvivoGen) was used asa positive control. The culture supernatant was collected 48 hourslater, and the amount of IFN-α produced in the culture supernatant wasassayed via ELISA.

<Results>

The results are shown in FIG. 9A and in FIG. 9B. FIG. 9A shows theresults concerning TLR2-knockout mice and TLR4-knockout mice, and FIG.9B shows the results concerning TLR7-knockout mice, TLR9-knockout mice,and MyD88-knockout mice. In the figures, “WT” represents the resultsconcerning wild-type mice. No changes in the capacity for IFN-αproduction of the Lactococcus lactis JCM5805 and JCM20101 strains wereobserved in TLR2- or TLR4-knockout mice, and the involvement thereof wasaccordingly denied. When either of the Lactococcus lactis JCM5805 orJCM20101 strain was added to mice, IFN-α in TLR9- and MyD88-knockoutmice completely disappeared by the knocking out of TLR9 and MyD88,respectively. Accordingly, TLR9 was verified to play a key role in IFN-αproduction by the Lactococcus lactis JCM5805 and JCM20101 strains.

Example 8 Identification of Active Substance

In Example 5, TLR9 was found to be a recognition receptor for theLactococcus lactis JCM5805 and JCM20101 strains. Identification of theligands thereof was attempted. DNA represented by CpG DNA is known as aTLR9 ligand. Concerning RNA, which is also a nucleic acid, ssRNArepresented by the RNA virus is known as TLR7L, and dsRNA is known asTLR3L. Since DNA or RNA was deduced to be a ligand, DNA and RNA wereextracted from both strains in order to inspect the activity.

<Experimental Method>

Preparation of DNA from Lactic Acid Bacteria

In accordance with the procedure of Example 1, Lactococcus lactisJCM5805, Lactococcus lactis JCM20101, and Lactobacillus rhamnosusATCC53103 were subjected to stationary culture. The strains wereharvested and then washed three times with sterile water. A solutionadjusted to comprise 50 mM Tris-HCl, 5 mM EDTA, and 6.7% sucrose (PH8.0) was added thereto. Subsequently, N-acetylmuramidase (2.5 mg/ml,Seikagaku Kogyo) and lysozyme (50 mg/ml, Seikagaku Kogyo) were added,and the resultant was allowed to stand at 37° C. for 45 minutes.Further, 50 mM Tris-HCl, 250 mM EDTA (PH 8.0), and 10% SDS were addedthereto, and the resultant was allowed to stand at 37° C. for 10minutes. 5.0 M NaCl was added, phenol, chloroform, and isoamyl alcohol(Wako Pure Chemicals Industries, Ltd.,) were added thereto, and themixture was subjected to centrifugation. The supernatant was selectivelyrecovered, ethanol in an amount twice the amount of the supernatant wasadded thereto, and the resultant was then subjected to centrifugation.The supernatant was removed, 70% ethanol was added to the precipitate,and centrifugation was then carried out. The supernatant was removed,RNase (Qiagen) was added thereto, and the resultant was allowed to standat 37° C. for 60 minutes. Further, 5.0 M NaCl was added thereto, phenol,chloroform, and isoamyl alcohol (Wako Pure Chemicals Industries, Ltd.,)were added thereto, and the mixture was subjected to centrifugation. Thesupernatant was selectively removed, ethanol in an amount twice theamount of the supernatant was added thereto, and the resultant was thensubjected to centrifugation. The supernatant was removed, 70% ethanolwas added to the precipitate, and centrifugation was then carried out.Nuclease Free Water (Qiagen) was added to the precipitate from which thesupernatant had been removed. DNAs of Lactococcus lactis JCM5805,Lactococcus lactis JCM20101, and Lactobacillus rhamnosus ATCC53103prepared in the manner described above were added to the pDC/mDC culturesystem at 0.1 μg/ml, 1 μg/ml, and 10 μg/ml, respectively. Thesupernatant was collected 48 hours later, and the amount of IFN-αproduced in the culture supernatant was assayed via ELISA. The bacterialstrains were used as controls.

Preparation of Total RNA from Lactic Acid Bacteria

In accordance with the procedure of Example 1, Lactococcus lactisJCM5805, Lactococcus lactis JCM20101, and Lactobacillus rhamnosusATCC53103 were subjected to stationary culture. The strains wereharvested and then washed three times with sterile water. RNAprotectBacteria Reagent (Qiagen) was added thereto, and the resultant wasallowed to stand at 37° C. for 5 minutes, followed by centrifugation.The supernatant was removed, lysozyme (5 mg/ml, Seikagaku Kogyo) wasadded, and the resultant was allowed to stand at 37° C. for 10 minutes.Thereafter, total RNAs were prepared from Lactococcus lactis JCM5805,Lactococcus lactis JCM20101, and Lactobacillus rhamnosus ATCC53103through DNase treatment (Qiagen) with the use of RNeasy Mini Kit(Qiagen). Total RNAs were added to the culture system according toExample 3 at 0.1 μg/ml, 1 μg/ml, and 10 μg/ml, respectively. The culturesupernatant was collected 48 hours later, and the amount of IFN-αproduced in the culture supernatant was assayed via ELISA. The bacterialstrains were used as controls.

<Results>

The results are shown in FIG. 10. DNAs of the Lactococcus lactis JCM5805and JCM20101 strains were found to have strong activity for inducingIFN-α, as expected. Activity was apparently detected when Lactococcuslactis JCM5805 was added at 1 μg/ml and when Lactococcus lactis JCM20101was added at 10 μg/ml. While no activity was detected in Lactobacillusrhamnosus ATCC53103, DNA thereof was found to exert activity at a levelequivalent to that of Lactococcus lactis JCM20101. More surprisingly,activity was detected when total RNA of Lactococcus lactis JCM20101 wasadded, and IFN-α production was induced at 1 μg/ml or higher.

These results demonstrate the following: (1) DNA is the active substanceof activity for inducing IFN-α production and DNA of an inactive strainhas activity; and (2) since lactic acid bacteria that activate pDCs andinduce IFN-α represented by the Lactococcus lactis JCM5805 and JCM20101strains are recognized by pDCs as strains, activity is detected withoutperforming DNA extraction. A strain such as Lactobacillus rhamnosusATCC53103 is inactive because it is not recognized by pDC; and (3) RNAof lactic acid bacteria becomes active besides DNA and it functions asTLRL, although it is very rare. If the examples mentioned above aretaken into consideration, RNA of Lactococcus lactis JCM20101 is the RNAligand for TLR9, the ligand of which has been known to be DNA,discovered for the first time.

Example 9

Examination of Effects of Ingestion of Lactococcus lactis JCM5805 UsingHealthy Mice

In the examples above, some lactic acid bacteria were found to have thecapacity for pDC activation and for induction of IFN-α production invitro. Thus, Lactococcus lactis JCM5805 was designated as arepresentative example and examined regarding the immunostimulatoryeffects in vivo attained via oral administration.

<Experimental Method>

Three groups of C57BL/6 mice (7-week-old, female) each consisting of 5individuals were provided: a group to which standard feeds (AlN93G,Oriental Yeast Co., Ltd.) are administered; a group to which mixed feedscontaining Lactococcus lactis JCM5805 are administered; and a group towhich mixed feeds containing Lactobacillus rhamnosus ATCC53103 areadministered. The dose of lactic acid bacteria was adjusted to 10 mg permouse per day. Blood sampling was carried out on day 0, day 3, and day 7(at the time of anatomy), and the amount of IFN-α produced in the bloodwas assayed via ELISA. At the time of anatomy, the spleen and themesenteric lymph node were extracted, and low-density cell fractionscontaining enriched dendritic cells were prepared in the mannerdescribed below. (In accordance with a conventional technique, spleniclymphocytes and mesenteric lymph node lymphocytes are prepared, thesecells are suspended in HBSS (Gibco) containing 20 mM HEPES (Gibco), andthe resulting cell suspension is superposed on 10%-FCS-containing RPMImedium (Sigma) comprising Histodenz (Sigma-Aldrich) dissolved to a finalconcentration of 15%. After centrifugation, cells in the intermediatelayer (i.e., low-density cell fractions) are recovered.) The low-densitycell fractions were stained with anti-CD11b-APC-Cy7 antibody (BDPharmingen), anti-mPDCA-1-APC antibody (Milteny Biotec), andanti-CD11c-PE-Cy7 antibody (eBiosciencs) for pDC gating and withanti-MHC class II-FITC antibody (eBiosciencs) and anti-CD86-PE antibody(eBiosciencs) as indicators for activation. The pDC gate(CD11c^(int)CD11b⁻mPDCA-1⁺) in vivo was determined via flow cytometry,and MHC class II and CD86 expression levels as pDC activation markerswere assayed. FIG. 11 shows a summary of a method for examining theeffects of Lactococcus lactis JCM5805 ingestion using healthy mice.

<Results>

FIG. 12 shows the results of blood IFN-α assays, and FIG. 13A to FIG.13D show the results of pDC activation. FIG. 13A and FIG. 13B showchanges in MHC class II levels in pDCs of the spleen and the mesentericlymph node, respectively, and FIG. 13C and FIG. 13D show changes in CD86levels in pDCs of the spleen and the mesenteric lymph node,respectively. The blood IFN-α level did not increase at all in the groupto which Lactobacillus rhamnosus ATCC53103 had been administered as inthe case of the group to which the standard feeds had been administered.In contrast, upward trends in the IFN-α levels were observed on day 3and day 7 in the group to which Lactococcus lactis JCM5805 had beenadministered (FIG. 12). Concerning pDC activation, changes in MHC classII or CD86 levels were not observed in either of the splenic ormesenteric lymph node lymphocytes of the group to which Lactobacillusrhamnosus ATCC53103 had been administered. While pDC activation did nottake place in the spleen of the group to which Lactococcus lactisJCM5805 had been administered, a significant level of activation wasobserved in both MHC class II and CD86 in the mesenteric lymph node.

The above results indicate that Lactococcus lactis JCM5805 wouldstimulate pDCs in vivo, as well as in vitro, and it would be capable ofinducing IFN-α production.

Example 10 Examination of Effects of JCM5805 Ingestion UsingImmunosuppression Models

The lactic acid bacteria according to the present invention would beadministered to persons with the weakened immune system or elderlypeople, as well as healthy persons. Thus, the effects of Lactococcuslactis JCM5805 ingestion were examined using immunosuppression models.

<Experimental Method>

Two groups of C57BL/6 mice (7-week-old, female) each consisting of 5individuals were provided: a group to which standard feeds (AlN93G,Oriental Yeast Co., Ltd.) are administered; and a group to which mixedfeeds containing Lactococcus lactis JCM5805 are administered. Theduration for administration of Lactococcus lactis JCM5805 was two weeks,the day on which administration of Lactococcus lactis JCM5805 wasinitiated was designated as “day −7,” and an immunosuppressive agent(Cyclophosphamide, Sigma-Aldrich) was administered intraperitoneally at200 mg/kg on day 0. Blood sampling was carried out on day −7, day −1,day 3, and day 7 (at the time of anatomy), and the blood IFN-α levelswere assayed in the same manner as in the examples above. Mice weresubjected to anatomy at the end and the degree of pDC activation in thespleen was assayed in the same manner as in the examples above. Bodyweights were measured simultaneously with blood sampling. FIG. 14 showsa summary of a method for evaluation of JCM5805 using immunosuppressionmodels.

<Results>

FIG. 15A shows changes in body weights of immunosuppression mouse modelsand FIG. 15B shows changes in blood IFN-α levels. FIG. 16A shows changesin MHC class II levels in pDCs and FIG. 16B shows changes in CD86 levelsin pDCs. FIG. 16C shows a percentage of pDCs and FIG. 16D shows theresults of flow cytometric analysis. A significant decrease was observedin body weights of mice of the group to which standard feeds had beenadministered after the administration of Cyclophosphamide. In contrast,body weight loss tended to be suppressed in the group to whichLactococcus lactis JCM5805 had been administered. In the group to whichstandard feeds had been administered, IFN-α became undetected in theblood at the end; however, IFN-α production was still observed at thetime of autopsy in the group to which Lactococcus lactis JCM5805 hadbeen administered. As a result of a comparison of the degree of pDCactivation in the spleen, MHC class II and CD86 levels significantlyincreased in the group to which Lactococcus lactis JCM5805 had beenadministered, compared with those in the group to which standard feedshad been administered. More interestingly, a percentage of pDCs in thespleen was significantly increased in the group to which Lactococcuslactis JCM5805 had been administered. The above results indicate thatorally-ingested Lactococcus lactis JCM5805 is able to antagonizeimmunosuppression induced by stress, aging, or other factors in dailylife, and the contribution of such strain to prevention of infectionscaused by a weakened immune system is significant.

Example 11 Verification of Proper Production of Dairy Products

With the use of the lactic acid bacteria according to the presentinvention, fermented milk (set yogurt, stirred yogurt) and naturalcheese were prepared.

The “set yogurt” is also referred to as a “firm yoghurt” or “stillyogurt,” which is subjected to fermentation in a container.

The “stirred yogurt” is also referred to as a “fermented yogurt” or“fluid yoghurt,” which is subjected to fermentation and then filled intoa container.

<Experimental Method> 1. Set Yogurt

(Mixed Culture with Lactobacillus bulgaricus and Streptococcusthermophilus)(1) As raw materials, raw milk (e.g., milk or skim milk powder),highly-branched cyclic dextrin (“Cluster Dextrin” (tradename), NihonShokuhin Kako Co., Ltd.), milk peptide (a general-purpose product), anda yogurt flavor (T. Hasegawa Co., Ltd.) were used.

FORMULATION TABLE 1 Composition ratio Milk 60% Skim milk powder 4.2%Milk peptide 0.10% Cluster Dextrin 1.0% Yogurt flavor 0.03% Lactic acidstarter (L. bulgaricus, St. thermophilus) 3.0% Lactic acid starter (Lc.lactis JCM5805) 3.0%(2) The raw materials were mixed to prepare a dispersion, the resultingdispersion was heated to about 70° C., and the resultant was applied toa homogenizer at a homogenization pressure (15 to 17 MPa). The resultantwas heat-sterilized at 95° C. for about 10 minutes, the resultant wascooled to about 35° C., lactic acid bacteria were added thereto(bacterial species: L. bulgaricus, St. thermophilus, and Lc. lactisJCM5805), and the resultant was filled into a container with a lid,followed by fermentation at 32° C. for about 6 to 7 hours. When theacidity of lactic acid reached 0.70, the resultant was cooled to 10° C.and stored.

Results:

(1) Flavor: good(2) Lactic acid bacteria count (Lc. lactis JCM5805): 10⁷ cells/g or more

2. Set Yogurt

(1) As raw materials, raw milk (e.g., milk or skim milk powder),highly-branched cyclic dextrin (“Cluster Dextrin” (tradename), NihonShokuhin Kako Co., Ltd.), milk peptide (a general-purpose product), anda yogurt flavor (T. Hasegawa Co., Ltd.) were used.

FORMULATION TABLE 2 Composition ratio Milk 60% Skim milk powder 4.2%Milk peptide 0.10% Cluster Dextrin 1.0% Yogurt flavor 0.03% Lactic acidstarter (Lc. lactis JCM5805) 6.0%(2) The raw materials were mixed to prepare a dispersion, the resultingdispersion was heated to about 70° C., and the resultant was applied toa homogenizer at a homogenization pressure (15 to 17 MPa). The resultantwas heat-sterilized at 95° C. for about 10 minutes, the resultant wascooled to about 35° C., lactic acid bacteria were added thereto(bacterial species: Lc. lactis JCM5805), and the resultant was filledinto a container with a lid, followed by fermentation at 32° C. forabout 16 hours. When the acidity of lactic acid reached 0.70, theresultant was cooled to 10° C. and stored.

Results:

(1) Flavor: good(2) Lactic acid bacteria count (Lc. lactis JCM5805): 10⁷ cells/g or more

3. Stirred Yogurt

(1) As raw materials, raw milk (e.g., milk or skim milk powder),highly-branched cyclic dextrin (“Cluster Dextrin” (tradename), NihonShokuhin Kako Co., Ltd.), milk peptide (a general-purpose product), anda yogurt flavor (T. Hasegawa Co., Ltd.) were used.

FORMULATION TABLE 3 Composition ratio Milk 60% Skim milk powder 4.2%Milk peptide 0.10% Cluster Dextrin 1.0% Yogurt flavor 0.03% Lactic acidstarter (Lc. lactis JCM5805) 4.0%

The raw materials were mixed to prepare a dispersion, the resultingdispersion was heated to about 70° C., and the resultant was applied toa homogenizer at a homogenization pressure (15 to 17 MPa). The resultantwas heat-sterilized at 125° C., the resultant was cooled to about 35°C., and lactic acid bacteria were added thereto (bacterial species: Lc.lactis JCM5805), followed by fermentation at 32° C. for about 16 hours.Fermentation was terminated at pH 4.6, the product was cooled to about20° C., and the resultant was filled into a container with stirring,followed by refrigeration at 10° C. or lower.

Results:

(1) Flavor: good(2) Lactic acid bacteria count (Lc. lactis JCM5805): 10⁷ cells/g or more

4. Drinkable Yogurt

(1) As raw materials, raw milk (e.g., milk or skim milk powder),highly-branched cyclic dextrin (“Cluster Dextrin” (tradename), NihonShokuhin Kako Co., Ltd.), milk peptide (a general-purpose product), anda yogurt flavor (T. Hasegawa Co., Ltd.) were used.

FORMULATION TABLE 4 Composition ratio Milk 30% Skim milk powder 6% Milkpeptide 0.20% Cluster Dextrin 1.0% Sugar 8% Yogurt flavor 0.03% Lacticacid starter (Lc. lactis JCM5805) 6.0%(2) The raw materials were mixed to prepare a dispersion, the resultingdispersion was heated to about 70° C., and the resultant was applied toa homogenizer at a homogenization pressure (15 to 17 MPa). The resultantwas heat-sterilized at 125° C., the resultant was cooled to about 35°C., and lactic acid bacteria were added thereto (bacterial species: Lc.lactis JCM5805), followed by fermentation at 32° C. for about 16 hours.Fermentation was terminated at pH 4.6, the product was cooled to about10° C., and the resultant was homogenized (in vacuo), followed byrefrigeration at 10° C. or lower.

Results:

(1) Flavor: good(2) Lactic acid bacteria count (Lc. lactis JCM5805): 10⁷ cells/g or more

5. Natural Cheese

(1) As raw materials, raw milk (i.e., milk), rennet (Standard Plus290,Christian Hansen), and calcium chloride (a general-purpose product) wereused.

FORMULATION TABLE 5 Raw materials Single strain Milk 100% Lactic acidstarter (Lc. lactis JCM5808) 3% Rennet 0.003% 20% calcium chloride0.0025% Whey removed −50%(2) Raw milk was heat-sterilized at 75° C. for 15 seconds, the resultantwas cooled to about 30° C., and lactic acid bacteria were added thereto(bacterial species: Lc. lactis JCM5805), followed by fermentation at 30°C. for about 1 hour. Fermentation was terminated at pH 6.4 and acidityof about 0.13, calcium chloride and rennet (Standard Plus290, ChristianHansen) were added, the mixture was stirred for about 3 minutes, andformation of curds was confirmed 30 minutes later. The curds were cutinto sizes of about 1 to 2 cm squares. The whey was removed, the curdswere packed into a mold, and the mold was inverted several times andallowed to stand for 12 hours.

The moisture content of the product was adjusted with pressurization byapplying the weight that is about 10 times greater than that of thecurds packed into the mold.

Results:

(1) Flavor: good(2) Lactic acid bacteria count (Lc. lactis JCM5805): 10⁷ cells/g or more

Example 12

Effects of Live Lactococcus lactis JCM5805, Lactococcus lactis 20101,and Lactobacillus rhamnosus ATCC53103

According to the examples above, activity of heat-killed bacteria isknown; however, whether or not live bacteria would act on pDCs remainsunknown. Thus, effects of live Lactococcus lactis JCM5805, Lactococcuslactis 20101, and Lactobacillus rhamnosus ATCC53103 on mouse pDCs wereinspected using the pDC/mDC culture system.

<Experimental Method> Preparation of Live Lactic Acid Bacteria

In accordance with the procedure of Example 1, Lactococcus lactisJCM5805, Lactococcus lactis JCM20101, and Lactobacillus rhamnosusATCC53103 were subjected to stationary culture. The strains wereharvested, washed three times with sterile water, and then suspended inPBS. The lactic acid bacteria count was determined using a particle sizedistribution measuring device (CDA-1000X, Sysmex Corporation), the cellswere added to the pDC/mDC culture system at concentrations of 1×10⁶,1×10⁷, and 1×10⁸ cells, and culture was conducted in a CO₂ incubator for48 hours. The culture supernatant was recovered, and the amount of IFN-αproduced in the culture supernatant was assayed.

<Results>

The results are shown in FIG. 18. While live Lactococcus lactis JCM5805and JCM20101 induced IFN-α production in a bacterial-count-dependentmanner, live Lactobacillus rhamnosus ATCC53103 did not induce IFN-αproduction at all. Regardless of whether or not the Lactococcus lactisJCM5805 and JCM20101 strains are heat-killed or alive, these strainswere found to activate pDCs and potently induce IFN-α production.

Example 13

Activity of Lactococcus lactis JCM5805 on Human pDC

In the examples above, lactic acid bacteria capable of acting on mousepDC were found; however, whether or not such bacteria were capable ofacting on human pDCs was unknown. Thus, pDCs were isolated from humanPBMCs with MACS, JCM5805 was designated as a representative sample, andactivity thereof on human pDCs was inspected.

<Experimental Method>

PBMCs were purchased from LONZA.

pDC Isolation with MACS and Purity Inspection

In accordance with the protocols of the Plasmacytoid Dendritic CellIsolation Kit (Miltenyi Biotec), human pDCs were isolated with MACS(purity: 97%). Human pDCs (5×10⁴ cells) were cultured on a 96-wellflat-bottom plate (Corning). To the isolated human pDCs, IL-3 (R&DSystems) was added at 10 ng/ml as the survival factor. In order toinspect the purity of human pDCs, human pDCs were stained withanti-CD123-FITC (AC145) antibody and anti-BDCA4-APC (AD-17F6) antibody(Miltenyi Biotec) for human pDC gating and then analyzed using the FACSCanto II (BD).

Addition of Ligand, Cell Culture, and ELISA

Lactococcus lactis JCM5805 was added to a final concentration of 10μg/ml, and culture was conducted in a CO₂ incubator for 24 hours. HumanIFN-α levels were assayed using the Human IFN-α ELISA Kit (PBLBiomedical Laboratories).

Analysis of IFN Gene Expression Via RT-PCR

The cultured cells were recovered, and total RNAs were extracted usingthe RNeasy Mini Kit (Qiagen). cDNA was synthesized from 200 ng of totalRNA using the iScript cDNA Synthesis Kit (Bio-Rad), and IFN-α1, IFN-β,IFN-λ1, and GAPDH genes were amplified via PCR using the synthesizedcDNA as a template. PCR was carried out using TaKaRa Ex Taq (TaKaRa) andthe primers described in Non-Patent Document 7. In accordance withgeneral protocols, IFN-α1, IFN-β, IFN-λ1, and GAPDH genes were subjectedto the reaction at 94° C. for 1 minute, and a cycle of 94° C. for 30seconds, at 49° C., 45° C., 49° C., and 45° C. for 30 seconds,respectively, and 72° C. for 15 seconds repeated 35 times, followed bythe reaction at 72° C. for 3 minutes. The PCR reaction solution waselectrophoresed in accordance with a general technique, and developmentof amplified fragments and the density thereof were inspected.

<Results>

The results are shown in FIG. 19. FIG. 19A, FIG. 19B, and FIG. 19C showthe purity of human pDCs isolated with MACS, the amount of IFN-αproduction detected at the protein level by ELISA, and IFN-α1, IFN-β,IFN-λ, and GAPDH gene expression levels detected by RT-PCR. With theaddition of Lactococcus lactis JCM5805, induction of IFN-α productionwas observed at the protein level. Also, induction of IFN-α1, IFN-β, andIFN-λ gene expression was detected. The above results demonstrate thatLactococcus lactis JCM5805 would also activate human pDCs.

Example 14

Influence of Yogurt Containing Lactococcus lactis JCM5805 on HumanAntiviral Activity

In the examples above, effects of Lactococcus lactis JCM5805 on mouseand human cells in vitro and effects thereof attained upon ingestion bymice were verified. On the basis of such results, effects attained uponingestion by humans were examined.

<Outline of Testing> Test Foods

Two types of test products described below were used.

(1) Test product: yogurt drink containing Lactococcus lactis JCM5805(2) Placebo product: yogurt-like drink containing no lactic acidbacteria

Purpose:

This test was carried out aimed at examination of the influence oflactic acid bacteria on blood biomarkers associated with antiviralactivity and on subjective evaluation by questionnaires concerningphysical conditions of healthy, working, adult males and females who hadcontinuously ingested yogurt drinks containing Lactococcus lactisJCM5805s for about 4 weeks, in comparison with the control experimentconducted with the use of yogurt-like drinks containing no lactic acidbacteria as placebos.

Test Subjects:

Test subjects were those who had no serious chronic disease, milkallergy, or other conditions, those who were evaluated to have noproblem by a particular virus test, those who were capable ofrestricting intake of yogurt and cheese during the period of testproduct ingestion, and those who were not on steroid medications(internal or external use).

Number of Subjects:

Thirty eight subjects (two groups each consisting of 19 subjects) wereemployed.

Test Design:

A randomized, double-blind, placebo-controlled, parallel-group study wascarried out.

Amount of Test Component to be Ingested Per Day:

A daily dose was about 1×10¹¹ cfu of Lactococcus lactis JCM5805.

Ingestion Method

Test subjects were asked to drink a bottle of the test product (100 ml)before or after meal in the morning every day.

Test Schedule

The period of test product ingestion was about 4 weeks. Blood samplingwas carried out three times: at a pre-test for grouping (1 month beforethe initiation of ingestion); at week 0 (the day before the initiationof ingestion); and at week 4 (the day after the termination ofingestion). Test subjects answered the questionnaires concerningphysical conditions every day during the ingestion period.

Evaluation Items

pDC activity in the blood (pDC surface markers: MHC class II and CD86),IFN-α gene expression in the blood, and the capacity for IFN-αproduction upon stimulation of peripheral blood mononuclear cells(PBMCs) with CpG DNA were assayed, and subjective evaluation of coldsymptoms was carried out in accordance with the questionnairesconcerning physical conditions.

<Experimental Method>

Blood biomarkers were analyzed by isolating PBMCs from the blood samplesobtained on week 0 and week 4 and examining the isolated PBMCs.

PBMCs (1×10⁶ cells) were stained with anti-CD123-FITC (AC145) (MiltenyiBiotec), anti-BDCA4-APC (AD-17F6) (Miltenyi Biotec), anti-CD86-PE (B7.2)(eBioscience), and anti-HLA-DR-PerCP (L243) (BD Biosciences) inaccordance with a conventional technique. HLA-DR (MHC class II) and CD86fluorescent intensities of the cell populations detected inCD123⁺/BDCA4⁺ were assayed with the use of FACS Canto II (BD), and thedetermined values were employed as the indicators for pDC activation.

IFN-α gene expression in the blood was detected by extracting total RNAsfrom 1×10⁶PBMCs using the RNeasy Mini Kit (Qiagen). cDNA was synthesizedfrom 100 ng of total RNA using the iScript cDNA Synthesis Kit (Bio-Rad),and the IFN-α1 gene (the GAPDH gene as a reference) was analyzed viareal-time PCR using the synthesized cDNA as a template. Real-time PCRanalysis was carried out using SYBR Premix Ex Taq (TaKaRa) and theprimers described in Non-Patent Document 7. In accordance with generalprotocols, the samples were subjected to the reaction at 95° C. for 10seconds, followed by a cycle of 95° C. for 10 seconds, 49° C. for 5seconds, and 72° C. for 10 seconds repeated 50 times.

In order to inspect the capacity for IFN-α production upon stimulationof the blood pDCs with CpG DNA, 5×10⁵ PBMCs were seeded on a 24-wellflat-bottom plate (Corning), and CpG-ODN2216 (CpG-A) (InvivoGen) wasadded thereto to the final concentration of 0.5 μM/ml. With respect toall the samples obtained from subjects, control samples containing noCpG DNA were prepared. Culture was conducted in a CO, incubator at 37°C. for 24 hours, the supernatant was recovered, and the amount of IFN-αproduction was assayed using the human IFN-α matched antibody pairs forELISA (eBioscience).

When Streptococcus pyogenes or influenza virus was allowed to act onhuman pDCs in vitro, the MHC class II expression level was elevated witha good response, although no significant increase was observed in theCD86 expression level (Non-Patent Document 8). Accordingly, MHC class IIwas designated as a major activation marker, and 36 samples exhibitingvalues of average MHC class II activity ±2SD (18 samples from eachgroup) were subjected to analyses of all biomarkers. The samples weredivided into those exhibiting values higher than the average MHC classII activity assayed at week 0 (hereafter referred to as “higher pDCactivity”) and those exhibiting values lower than such average(hereafter referred to as “lower pDC activity”), and these samples wereseparately analyzed.

In the questionnaires concerning physical conditions, 7 main coldsymptoms (i.e., runny nose, stuffy nose, sneezing, sore throat, itchythroat, coughing, headache, and fever) were rated using a 5-point scale(from 1: no symptom, to 5: severe symptom) every day. The average of the7 items was designated as the indicator for the severity of coldsymptoms.

<Results>

The results of assays for pDC activity in the blood are shown in FIG.20. FIG. 20A and FIG. 20D show changes in MHC class II and CD86 activityin pDCs observed in the activity assays from week 0 to week 4. Changesin MHC class II and CD86 activity of the group that had ingested yogurtdrinks containing Lactococcus lactis JCM5805 (hereafter, referred to as“the JCM5805 group”) were significantly higher than those of the groupthat had ingested yogurt-like drink containing no lactic acid bacteria(hereafter, referred to as “the placebo group”). The results of analysesseparately conducted for the samples exhibiting higher pDC activity andfor the samples exhibiting lower pDC activity and the changes in MHCclass II activity are shown in FIG. 20B and FIG. 20C, respectively. Theresults as mentioned above and the changes in CD86 activity are shown inFIG. 20E and FIG. 20F, respectively. Regarding changes in MHC class IIactivity, there was no significant difference in samples exhibitinghigher pDC activity between the JCM5805 group and the placebo group. Inthe case of samples exhibiting lower pDC activity, however, such changesof the JCM5805 group were significantly higher than those of the placebogroup. Regarding changes in CD86 activity, no significant difference wasobserved between the JCM5805 group and the placebo group, regardless ofpDC activity levels. This is considered to occur because CD86 is lesslikely to be influenced by pDC activity, as described in Non-PatentDocument 8. The above results demonstrate that pDC activity is elevatedupon ingestion of Lactococcus lactis JCM5805 and that the effects aremore significant for subjects exhibiting lower pDC activity and having aweaker immune system.

FIG. 21 shows the results of analysis of IFN-α gene expression in theblood at lower pDC activity. At lower pDC activity, no significantchanges were observed from week 0 to week 4 in the placebo group;however, a significant increase was observed in expression levels fromweek 0 to week 4 in the JCM5805 group. At higher pDC activity, nosignificant changes were observed from week 0 to week 4 in the placebogroup and in the JCM5805 group (data not shown). The results demonstratethat the amount of IFN-α gene transcription in the human blood isincreased by ingestion of Lactococcus lactis JCM5805.

FIG. 22 shows the results of assays for the capacity for IFN-αproduction attained when the blood pDCs exhibiting lower pDC activityare stimulated with CpG DNA. At lower pDC activity, no significantchanges were observed from week 0 to week 4 in the placebo group;however, a significant increase was observed from week 0 to week 4 inthe JCM5805 group. At higher pDC activity, no significant changes wereobserved from week 0 to week 4 in the JCM5805 group (data not shown).CpG DNA is a nucleic acid ligand targeting TLR9, and the virusrecognition mechanism of pDC detects viral DNA or RNA by means of TLR9or TLR7/8. Accordingly, the virus recognition mechanism was wronglystimulated by addition of a nucleic acid ligand (i.e., CpG DNA).Specifically, the results of the experiments indicate that pDCactivation induced by CpG DNA stimulation is potentiated in the JCM5805group and it leads to an enhanced response at the time of virusinfection.

FIG. 23 shows the results of the questionnaires concerning physicalconditions. For the JCM5805 group and the placebo group, the totalnumber of days during which cold symptoms had developed and the totalnumber of days during which cold symptoms did not develop weredetermined in every week, and the outcomes were subjected to the squaretest. As a result, the total number of days during which the subjects inthe JCM5805 group had developed cold symptoms was found to besignificantly fewer, and the total number of days during which thesubjects did not develop cold symptoms was found to be larger on week 4,compared with the placebo group. This indicates that continuousingestion of Lactococcus lactis JCM5805 for 4 weeks leads the subjectsto be less susceptible to colds.

The above results demonstrate that blood pDCs are activated when humaningests Lactococcus lactis JCM5805, the capacity for IFN-α production isenhanced, and responses upon virus infection are improved, which wouldconsequently lead a person to be less susceptible to colds. Such effectswere particularly significant for subjects with lower immunity againstvirus infection (pDC activity) and at a high risk of catching a cold.

All publications, patents, and patent applications cited herein areincorporated herein by reference in their entirety.

INDUSTRIAL APPLICABILITY

Lactic acid bacteria capable of activating pDCs and inducing IFNproduction can be used for an immunostimulatory pharmaceutical productor food or drink product as the agent for inducing IFN production.

1. An agent for inducing IFN production comprising, as an activeingredient, lactic acid bacteria capable of activating plasmacytoiddendritic cells (pDCs) and inducing IFN production or a cultured orprocessed product thereof.
 2. The agent for inducing IFN productionaccording to claim 1, wherein the processed product of lactic acidbacteria is a fraction containing nucleic acids.
 3. The agent forinducing IFN production according to claim 1, wherein IFN is type I IFNor type III IFN.
 4. The agent for inducing IFN production according toclaim 1, wherein IFN is at least one member selected from the groupconsisting of IFN-α, IFN-β, and IFN-λ.
 5. The agent for inducing IFNproduction according to claim 1, wherein, when the agent is orallyadministered, the lactic acid bacteria capable of activatingplasmacytoid dendritic cells (pDCs) and inducing IFN production arehighly tolerant to the gastric juice or intestinal juice and are capableof reaching the intestinal canal alive.
 6. The agent for inducing IFNproduction according to claim 5, wherein the lactic acid bacteriacapable of activating plasmacytoid dendritic cells (pDCs) and inducingIFN production are Lactococcus lactis JCM5805.
 7. An immunopotentiatingagent comprising the agent for inducing IFN production according toclaim
 1. 8. An agent for prevention or treatment of virus infectioncomprising the agent for inducing IFN production according to claim 1.9. The immunopotentiating agent according to claim 7, which is an oralpreparation.
 10. The agent for prevention or treatment of virusinfection according to claim 8, which is an oral preparation.
 11. A foodor drink product comprising the agent for inducing IFN productionaccording to claim
 1. 12. The food or drink product according to claim11, which is a dairy product containing cheese or yogurt.
 13. A methodof screening for lactic acid bacteria capable of activating plasmacytoiddendritic cells (pDCs) and inducing IFN production comprising culturingthe lactic acid bacteria with bone marrow cells and detecting activationof plasmacytoid dendritic cells (pDCs) and induction of IFN production,wherein, when plasmacytoid dendritic cells (pDCs) are activated and IFNproduction is induced, the lactic acid bacteria are determined to becapable of activating plasmacytoid dendritic cells (pDCs) and inducingIFN production.
 14. A host microorganism for a recombinant vaccinecomprising the agent for inducing IFN production according to claim 1.